CANCER GENOMICS & PROTEOMICS 14 : 299-313 (2017) doi:10.21873/cgp.20041

Review

Histone Deacetylases as New Therapeutic Targets in Triple- negative Breast Cancer: Progress and Promises NIKOLAOS GARMPIS 1* , CHRISTOS DAMASKOS 1,2* , ANNA GARMPI 3* , EMMANOUIL KALAMPOKAS 4, THEODOROS KALAMPOKAS 5, ELEFTHERIOS SPARTALIS 2, AFRODITE DASKALOPOULOU 2, SERENA VALSAMI 6, MICHAEL KONTOS 7, AFRODITI NONNI 8, KONSTANTINOS KONTZOGLOU 1, DESPINA PERREA 2, NIKOLAOS NIKITEAS 2 and DIMITRIOS DIMITROULIS 1

1Second Department of Propedeutic Surgery, Laiko General Hospital, National and Kapodistrian University of Athens, Medical School, Athens, Greece; 2N.S. Christeas Laboratory of Experimental Surgery and Surgical Research, Medical School, National and Kapodistrian University of Athens, Athens, Greece; 3Internal Medicine Department, Laiko General Hospital, University of Athens Medical School, Athens, Greece; 4Gynaecological Oncology Department, University of Aberdeen, Aberdeen, U.K.; 5Assisted Conception Unit, Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; 6Blood Transfusion Department, Aretaieion Hospital, Medical School, National and Kapodistrian Athens University, Athens, Greece; 7First Department of Surgery, Laiko General Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; 8First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

Abstract. Triple-negative breast cancer (TNBC) lacks therapies. Given the fact that epigenetic processes control both expression of estrogen receptor (ER), progesterone receptor the initiation and progression of TNBC, there is an increasing (PR) and HER2 gene. It comprises approximately 15-20% of interest in the mechanisms, molecules and signaling pathways breast cancers (BCs). Unfortunately, TNBC’s treatment that participate at the epigenetic modulation of genes continues to be a clinical problem because of its relatively expressed in carcinogenesis. The acetylation of histone poor prognosis, its aggressiveness and the lack of targeted proteins provokes the transcription of genes involved in cell therapies, leaving chemotherapy as the mainstay of treatment. growth, and the expression of histone deacetylases (HDACs) It is essential to find new therapies against TNBC, in order to is frequently up-regulated in many malignancies. surpass the resistance and the invasiveness of already existing Unfortunately, in the field of BC, HDAC inhibitors have shown limited effect as single agents. Nevertheless, their use in combination with kinase inhibitors, autophagy inhibitors, This article is freely accessible online. ionizing radiation, or two HDAC inhibitors together is currently being evaluated. HDAC inhibitors such as *These Authors contributed equally to this study. suberoylanilidehydroxamic acid (SAHA), sodium butyrate, mocetinostat, panobinostat, entinostat, YCW1 and N-(2- Correspondence to: Nikolaos Garmpis, MD, Second Department of hydroxyphenyl)-2-propylpentanamide have shown promising Propedeutic Surgery, Laiko General Hospital, National and therapeutic outcomes against TNBC, especially when they are Kapodistrian University of Athens, Medical School, 17 Agiou used in combination with other anticancer agents. More Thoma Street, 11527, Athens, Greece. Tel: +30 6976291403, Fax: +30 2132061772, e-mail: [email protected] studies concerning HDAC inhibitors in breast carcinomas along with a more accurate understanding of the TNBC’s Key Words: Histone deacetylase, HDAC inhibitors, breast cancer, pathobiology are required for the possible identification of triple negative, epigenetics, review. new therapeutic strategies.

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According to the World Health Organization, breast cancer Table I. Classification of breast cancers and their relation to breast (BC) is the most frequently diagnosed cancer and the second cancer cell lines. leading cause of cancer-related death, among women Classification Immuno-profile Cell line worldwide. Interestingly, according to the American Cancer Society about 12% of women in U.S.A. will develop BC Luminal A ER+, PR±, HER2- MCF-7, T47D during their lifetime (1-4). There is a variety of risk factors Luminal B ER+, PR±, HER2+ PR BT-474, ZR-75 that are associated with BC such as gender, age, ethnicity or HER2+ ER –, PR –, HER2+ MDA-MB-453 TNBC (Basal-like) ER –, PR –, HER2 – BT-20, MDA-MB-468 lifestyle, but it is now clear that the most dangerous factor for TNBC (Claudin-low) ER –, PR –, HER2 – BT549, Hs578T, the development of BC is heredity. In particular, in high-risk MDA-MB-231 families, there have been reported more than 1.000 mutations of breast cancer 1 ( BRCA1 ) and breast cancer 2 ( BRCA2 ) TNBC: Triple-negative breast cancer; ER: estrogen receptor; PR: genes (1). progesteron receptor. BC is a very heterogeneous disease given that its invasive process is associated with a variety of molecular alterations. These changes lead to different subtypes of the disease. The HDACs and their Μechanism of Αction current classification divides BC subtypes into Luminal A, Luminal B, HER2-positive and TNBC (5, 6). A better Today, several BC subtypes are treated with many different explanation of BC classification and the corresponding cell primary therapeutic protocols, but none of them uses lines are presented in Table I (7, 8). epigenetic drugs, despite the fact that the interest for First of all, Luminal A and B represent from 65% to 80% of epigenetics in BC tumorgenesis is increasing (12, 13). total BC disease. More specifically they account 50-60% and Gene regulation is affected by nucleosome packaging. 15-20% respectively (9). These subtypes are generally Nucleosome is the organizing DNA structure that is consisted correlated to a good prognosis. They are known to express the of about a 200 bp DNA coiled around an octameric core, estrogen receptor (ER-positive) that participates in cell consisted of histone proteins (14). proliferation, viability, and invasion of BC cells. We should N-terminal tails of histones extend outward from the mention that patients with luminal B tumors frequently present nucleosomal core and are modified by the covalent addition a worse prognosis than Luminal A patients. This could be of groups to the side chains of certain amino acids. These explained as in Luminal B tumors HER2 gene, which is modifications, including methylation, acetylation, associated with potent proliferation, can be amplified (10). The phosphorylation and the reverse processes, can change the substantial difference though, between Luminal A and B secondary DNA structure. As a result, gene promoter regions patients is the cell proliferation rate which is higher in the latter. become accessible or inaccessible to transcription factors (15). Secondly, there is the subtype of HER2-positive patients Histone acetyltransferases (HATs) catalyze the reversible who amplify the oncogene HER2 . These tumors are ER process of lysine acetylation at the ε- amino group of negative, so they are different from Luminal B cancers. proteinogenic lysine residues. Histone acetylation neutralizes Unfortunately, hormonotherapy is inefficient against HER2- the positive charge of lysine residues, therefore is correlated positive tumors. During the last 20 years the development to chromatin relaxation and active gene transcription (17). On of novel drugs targeting HER2 ( e.g. trastuzumab, lapatinib, the other hand, functional antagonists of HATs, histone pertuzumab) has enhanced the clinical outcomes (1). deacetylases (HDACs) remove the acetyl groups (16), thus Moreover, another severe subtype of BC is triple negative leading to compressed chromatin structure (heterochromatin), breast cancer (TNBC). It is associated with poor prognosis. and subsequently suppressing gene transcription (18) (Figure TNBC does not express either estrogen, or progesterone 1). Except for the direct effect of acetylation on chromatin receptors, or HER2 gene. These tumors can be further structure, gene regulation through acetylation is based on classified in several subtypes. The first subgroup is basal-like, synergistic actions. The specific acetylation patterns on histone where tumors express some characteristics of breast tails recruit further chromatin modulators that form co- myoepithelial cells. Basal-like tumors are highly proliferative repressor or co-activator complexes. and are associated with very poor prognosis. Another Several human HDACs have been identified; recently subgroup is claudin-low, which presents epithelial to HDACs have been classified into four classes in accordance mesenchymal transition (EMT) and stem cell-like or/and to functional criteria and homology to yeast proteins (19). In tumor initiating cell features (11). This subtype is also general, HDACs can be divided into Zn 2+ -dependent classes associated with poor prognosis. (class I, II and IV) and NAD-dependent classes (class III). Neoadjuvant anthracycline/taxane-based chemotherapies Class I is consisted of HDACs 1, 2, 3 and 8. Class II can be (e.g. , docetaxel, doxorubicin, cyclophosphamide) have been divided further into class IIa (HDAC4, 5, 7, and 9) and class tested in TNBC patients with poor prognosis (5). IIb (HDAC6 and 10). Class III members, or sirtuins as they

300 Garmpis et al : Histone Deacetylase Inhibitors in Triple Negative Breast Cancer (Review)

Figure 1. Therapeutic strategy targeting histone deacetylases against triple-negative breast cancer and histone deacetylase inhibitors classification. TNBC: Triple-negative breast cancer; HAT: histone acetyltransferases; HDAC: histone deacetylases.

are often called since they are homologous to silent Clinical trials (phases I and II) have also demonstrated that information regulator 2 (SIR2) of Saccharomyces cerevisiae , HDAC inhibitors result in minor adverse effects in patients consist of SIRT1-SIRT7. Finally, class IV has only one (15, 26-30). Their mechanism of action involves binding of member, HDAC11 (20) (Figure 1). their hydroxamate group to the zinc cation (Zn 2+ ) located in the HDAC cavity (31). Several clinical trials seemed to have HDAC Inhibitors as Anti-cancer Agents a beneficial result. For instance, the US Food and Drug Administration has approved SAHA and romidepsin as HDAC inhibitors can be classified into four classes according regimen of cutaneous T-cell lymphoma (29, 31) and peripheral to their chemical structure: a) hydroxamates ( e.g. T-cell lymphoma (27) respectively. Moreover, panobinostat suberanilohydroxamic acid (SAHA)), b) benzamides ( e.g. MS- treatment is reported to be clinically successful against 275), c) cyclic peptides ( e.g. romidepsin) and d) aliphatic acids multiple myeloma (28). (e.g. valproic acid). Alternatively, HDAC inhibitors can be In total, advanced stages of clinical trials have studied classified according to their specificity for HDAC subtypes or several HDAC inhibitors against many cancer types. However, classes. For example, SAHA and trichostatin A are pan-HDAC concerning the TNBC field, studies have shown that, in inhibitors, while MS-275 and romidepsin inhibit class I and general, HDAC inhibitors succeed clinically beneficial results valproic acid inhibits class I and IIa HDACs (21). as complementary treatment ( e.g. SAHA and VPA), or in It is clear that both histone acetylation and deacetylation combination with cytotoxic drugs and ionizing radiation affect chromatin remodeling as strong epigenetic (Table II). mechanisms. Interestingly, evidence from several reports indicates that HDAC levels are increased in certain cancer SAHA. SAHA or vorinostat is a pan-HDAC inhibitor that types (22-24). In addition, HDAC inhibitors have been induces apoptosis in several types of haematological and solid reported to enhance the acetylation of histones, in tumor tumor cells (32). Clinical investigations show that SAHA is a cells (25). Unlike other cytostatic-type compounds, HDAC potent inhibitor against tumor types at doses that were well inhibitors have been reported to exert much lower cytoxicity tolerated by patients (33). on normal cells, than on cancer cells. In general, HDAC According to a study in human TNBC cell lines, MDA- inhibitors induce the inhibition of tumor growth and the MB-231 and inBT-549, SAHA significantly promotes in vitro apoptosis of cancer cells. motility via activation of epithelial–mesenchymal transition

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Table II. Histone deacetylase inhibitors and their action against triple-negative breast cancer.

HDAC inhibitors Co-treatment Action

SAHA Alone Promotion of EMT via HDAC8/FOXA1 signals Olaparid Inhibition of tumor growth, apoptotic + autophagic cell death IR Inhibition of tumor growth, DNA damage Parthenolide Nucleosome conformation, activation of caspase-3, relegation of beclin-1, inhibition of autophagy Sorafenib Apoptotic + autophagic cell death Cisplatin Inhibition of HDAC6, apoptosis, cell cycle arrest Ferrocifen Anti-proliferative activity NaB Anti-proliferative activity, cell cycle arrest at G 0/G 1 phase, apoptosis, decrease of phosphorylation, protein and mRNA expression of wtp53 NaB TSA G6PDH activation, rising of oxygen consumption Mocetinostat Alone Inhibition of genes involved in cell cycle regulation JQ1 Decrease of cell viability, increase in the expression of USP17L5,decrease of Ras activity Panobinostat Alone Apoptosis, anti-proliferative activity, inhibition of primary tumor volumes, increase of CDH1, cell cycle arrest at G 2/M phase, restoration of ERa mRNA+ protein expression, anti-EMT effects Salinomycin Anti-proliferative activity, inhibition of breast cancer stem cell properties, suppression of ALDH1 activity Chloroquine Inhibition of tumor growth, increase in viability Entinostat Alone Anti-EMT effects, increase of E-cadherin, inhibition of HDAC1, inhibition of cell migration, prevention of metastasis ATRA+Doxorubicin Inhibition of tumor growth, inhibition of Topo II- β, silence of RAR- β YCW1 IR Autophagic cell death, inhibition of BNIP3 Compound 2 Alone Anti-proliferative activity, target HDAC8

HDAC: Histone deacetylases; SAHA: suberoylanilidehydroxamic acid; NaB: sodium butyrate; YCW1: [3-(2-(5-methoxy-1H-indol- 1yl)ethoxy)phenyl]-amide N-hydroxyamide; IR: ionizing radiation; TSA: trichostatin A; ATRA: all-trans retinoic acid; EMT: epithelial to mesenchymal transition; FOXA1; forkhead-box protein A1; G6PDH: glucose-6phosphate dehydrogenase; CDH1: cadherin; ERa: estrogen receptor a; ALDH1: aldehyde dehydrogenase 1; TopoII- β: topoisomerase II- β; RAR- β: retinoic acid receptor; BNIP3: BCL2/adenovirus E1B 19 kDa protein- interacting protein 3.

(EMT) phenotype (34). This phenotype is known as the first silencing of HDAC8, but not HDAC6 (34). Previous studies and critical point for metastatic dissemination of cancer cells. have suggested that HDAC6 and HDAC8 mediate the SAHA Vimentin up-regulation and E-cadherin down-regulation are effects in BC cells (39, 40). HDAC8 lacks the conserved C- considered as markers of EMT (35, 36). terminal domain (41), so it is a unique class I HDAC and it Wu et al. found that SAHA treatment induces the has a different expression profile from those of HDACs 1-3 expression of the mesenchymal markers N-Cadherin, Vimentin (42). HDAC8 silencing does not change histone acetylation and Fibronectin, while decreases the expression of epithelial (43), but it has been assumed that it could act at certain marker E-Cadherin. Nevertheless, SAHA treatment does not promoter sites of FOXA1 . Based on this evidence, researchers change neither the expression nor the nuclear translocation of claimed that HDAC8/FOXA1 signals motivated by SAHA transcription factors related to EMT. These factors were zinc treatment induce the EMT of TNBC cells (34). finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG), basic As it is known, HDACs participate at the homologous helix-loop-helix transcription factor Twist-related protein recombination repair pathway of DNA, given the fact that they (TWIST) and Zinc finger E-box-binding homeobox (ZEB). As regulate the expression of genes related to this procedure (15). a result it seemed that these epithelial–mesenchymal transition Studies have shown that ADP-ribose (poly) polymerase related transcription factors are not involved in this process. (PARP) inhibitors, such as olaparid, could be used as anti- Moreover, SAHA treatment decreased mRNA and protein cancer treatment). Min et al. have searched whether SAHA expression of forkhead-box protein A 1 (FOXA1) (34). treatment enhances the anti-tumor effect of poly (ADP-ribose) FOXA1, as a growth factor, mediates the hormonal response polymerase (PARP) inhibitors in TNBC cells by blocking the in BC (37, 38). When FOXA1 is over-expressed, SAHA homologous recombination repair pathway (46). Investigators induced EMT of TNBC cells is limited. used MDA-MB-157, -231, -453, -468 and BT-549 human cells Furthermore, in MDA-MB-231 and BT-549 cells, SAHA- and found that combinational treatment with olaparid and induced down-regulation of FOXA1 transcription and up- SAHA suppressed the proliferative signaling pathway and regulation of N-cadherin and vimentin were attenuated by promoted inhibition of tumor growth, in comparison to

302 Garmpis et al : Histone Deacetylase Inhibitors in Triple Negative Breast Cancer (Review) olaparid or SAHA treatment alone. An in vivo study of a terminal tails of histones H3 and H4 and these findings were MDA-MB-231 xenograft model confirmed this evidence. in accordance to other studies (63, 64). Furthermore, Moreover, this combination seemed to induce both apoptotic investigators claimed that there is an association between this and autophagic cell death, thus enhanced the cytotoxic effects histone hyperacetylation provoked by SAHA or SAHA and of these inhibitors (46). Further studies could examine more PN treatment and the up-regulation of tumor suppressor combined treatments of PARP inhibitors with other HDAC factors p21 and p27, as well as down-regulation of inhibitors in order to improve therapeutic approaches for fundamental survival proteins Bcl-2 (55). These results have TNBC patients. also been confirmed by another study (65). This evidence Furthermore, it is known that the majority of deaths in demonstrates that epigenetic actions could induce the TNBC patients are not due to the primary tumors, but to alterations in the expression of these proteins. metastases. An in vitro study on MDA-MB-231 and 4T1 (a Investigators have also studied the production of reactive mouse TNBC cell line) cell lines for the investigation of the oxygen species (ROS) and the autophagic process and they effects of ionizing radiation (IR) and SAHA co-treatment, have found that SAHA treatment urges both processes. Both indicated that the combination was therapeutically more ROS generation and autophagy are reduced by two NADPH effective in a statistically significant way, than IR or SAHA oxidase family (NOX) inhibitors - Nox enzymes are major alone. Tumor growth was also inhibited, in vivo , in an ROS producers-, apocynin and diphenyleneiodonium chloride orthotopic BC mouse model (47). In addition, this combination (DPI). Therefore, it was proposed that SAHA primarily increased DNA damage through the inhibition of DNA repair induced NOX to increase the production of ROS, which led proteins. Concerning SAHA treatment in 4T1 cells, it led to to activation of autophagy (55). Another study has shown that inhibition of BC cell migration and invasion, via the inhibition SAHA stimulates autophagy in tumor cells (66). When they of matrix metalloproteinase-9 (MMP-9) activity. added PN, SAHA’s results on ROS generation and autophagic In general, evidence shows, in several cancer types, MMPs process were diminished, while they observed a potent mediate the invasion and metastasis (48). MMP-9 participates activation of apoptosis. at the degradation of type IV collagen and its over-expression Finally, it has been found that SAHA and PN combined has been found to be associated with invasion and metastatic therapy induced a decrease of the expression levels of beclin-1, potential in many types of carcinomas (49-53). In total, a protein involved in autophagosome formation (55). Previous evidence indicates that SAHA could serve as a radiosensitizer studies have shown that beclin-1 is cleaved by caspases (67). against TNBC (47). N-terminal cleavage fragment of beclin-1 suppresses autophagy, Also, in the same study, an in vivo experimental metastasis while its C-terminal cleavage fragment promotes the release of mouse model was used and showed that SAHA treatment cytochrome C from mitochondria (68). As an extension to the inhibited lung metastasis and enhanced radiosensitivity (47). above mentioned finding, investigators have claimed that Concerning metastases, another study of an in vivo model of SAHA/PN combination treatment relegates beclin-1 by TNBC has reported that SAHA treatment is involved in the activating caspase-3. This event could inhibit SAHA- induced induction of DNA double-strand breaks suppressing brain autophagy and the release of cytochrome C (55). Taken together metastatic colonization by 62% (54). Further studies that this evidence, it could be implied that SAHA/PN co-treatment combine SAHA with DNA active drugs or radiation are could be a candidate for TNBC therapy. needed in order to investigate thoroughly their actions against A recent study on MDA-MB-231 cells used SAHA in metastases. comparison to a kinase inhibitor and found that this co- Carlisi et al. studied the combination of SAHA and treatment induces cell death. An important fact was that cell parthenolide (PN) (55). The principal bioactive sesquiterpene death was induced by both autophagy and apoptosis (69). lactone component of feverfew Tanacetum parthenium , PN, These results were confirmed by Zhang et al. , who used was found to induce tumor inhibition on many cancer types, SAHA and the antiangiogenic kinase inhibitor Sorafenib in such as osteosarcoma, melanoma (56) and prostatic (57), epithelial tumor cell types and found that this treatment pancreatic (58), colorectal (59) cancers. At the same time it suppressed the expression of anti-apoptotic proteins and showed low toxicity against normal cells. PN acts increased the activation of CD95 extrinsic apoptotic and cytotoxically on MDA-MB231 cells, but at low doses it is not lysosomal protease pathways. So this combination has been effective (60, 61). Investigators have combined PN and SAHA found to interact synergistically against liver, kidney, and in order to overcome the PN’s deficiency (55). pancreatic tumor cells in vitro (70). These studies indicate that They claimed that pre-treatment of MDA-MB231 cells with the combination of a kinase and an HDAC inhibitor is a SAHA sensitized the cells to the cytotoxic effect of PN (62), promising treatment modality for the prevention or therapy of so PN and SAHA acted synergistically in order to fight against aggressive cancer types, especially TNBC. TNBC cells (55). Moreover, they demonstrated that SAHA The effects of HDAC inhibitors (VPA and SAHA) and treatment leads to acetylation of lysine residues in the NH 2- cisplatin (CDDP), a chemotherapeutic agent, co-treatment

303 CANCER GENOMICS & PROTEOMICS 14 : 299-313 (2017) against several types of BC cells have been explored using an protein levels of mutant p53 (mtp53), while neither of these isobolographic method (71,72). This combination achieved to compounds had anti-proliferative effects on wild type p53 reduce the doses of the compounds and to be therapeutically (wtp53) via time and dose dependent manners (84). p53 gene beneficial for head and neck squamous cell carcinomas (26, has been reported as the most frequently mutated gene in BC 73) and BC in vitro (74, 75). This study also showed that in (85). Approximately, 62% of the basal-like TNBC and 43% of MDA-MB231 cells, the combination of the CDDP and VPA the non-basal-like TNBC have mutations in the mtp53 (86). at the ratio of 1:1 induced antagonistic interactions, while These data urged the investigators to report that the CDDP and SAHA co-treatment induced synergistic inhibition occurs during the transcription, given that the interactions (71). According to the investigators, this could be transcription of the precursor p53 is down-regulated more explained by the fact that SAHA is a potent inhibitor of the rapidly (less than 2h) and aggressively than that of mature HDAC6 activity compared to VPA (26). Many nuclear p53. Also, the transcription of mtp53 is inhibited by the proteins, like the transcription factor forkhead boxp3 (FOXP3) silencing of HDAC8, while not of HDAC6 (84). Both and the DNA repair factor KU70, are substrates of HDAC6, HDAC6 and HDAC8 are previously reported to mediate the which is recruited by RNA polymerase II to chromatin in down-regulation of p53 induced by HDAC inhibitors (40, 87). order to suppress the cycles of acetylation/deacetylation. This Moreover, both SAHA and NaB reduced the binding of procedure enables the transcription and transfers cytotoxic transcription factor Yin Yang 1 (YY1) with the -102 to -96 poly-ubiquitinylated proteins into autophagosomes (76). All position of human p53 promoter. Interestingly, silencing of these mechanisms of HDAC6 action and its potential YY1 significantly inhibits the mtp53 transcription (83). YY1 inhibition by SAHA could explain the hypothesis. is suggested to be over-expressed in BC cells and to be a key Moreover, it has been reported that CDDP/HDAC inhibitor molecule in BC progression. Knockdown of YY1 led to co-treatment induces apoptosis and cell-cycle arrest of TNBC suppression of the clonogenicity, migration, invasion and cells (71). Further research in vivo is necessary in order to use tumor formation of BC cells (88). this promising combination therapy in TNBC clinical trials. Based on the above, researchers have found that both A recent study has described a new family of hybrid SAHA and NaB reduced the association of HDAC8 and YY1. compounds that combine particular structural motifs of As a result, acetylation of residues 170–200 of YY1 was tamoxifen (TAM), ferrocifen (FcTAM) and SAHA (77). enhanced, leading to decreased YY1 transcription and finally, Ferrocenyl group (Fc) is reported to act as a redox antenna for inhibition of the YY1- induced p53 transcription (84). In total, phenol oxidation via an intra-molecular mechanism (78-80). this study showed that not only SAHA and NaB treatment They have found that ferrocene derivatives are extremely could be very promising for TNBC patients, but also more active than organic analogs in MDA-MB-231 cells and HDAC8/YY1/mtp53 signals could become an important target hormone-dependent MCF-7 BC cell lines. FcTAM improved for TNBC therapy. its inhibitory activity by replacing its 3-(dimethylamino) propan-1oxy group with an 8-hydroxyamino-8-oxooctanamido Trichostatin A and NaB. Trichostatin A (TSA) is an antifungal or an 8-amino-8-oxooctanamido group. In MDA-MB-231 BC antibiotic, found in cultured mammalian cells and, at low cells, FcTAM–SAHA treatment exerted better anti- nanomolar concentrations, in fractionated cell nuclear extracts. proliferative effects than SAHA treatment alone (77). In general, as an HDAC inhibitor, TSA not only suppresses Interestingly, the organometallic compounds induced the HDAC activity, but also leads to cell-cycle arrest in G 1 and expression of p21waf1/cip1 gene in MCF-7 cells in G2 phase, induces cell differentiation as well as the reversion accordance with their antiproliferative activity (77). Further of transformed cells in vitro (89). research on the field of organometallic moiety could A study of the effect of NaB and TSA on viability of non- contribute to a more proper treatment against TNBC. tumorgenic MCF10A cells and of MCF-7, T-47D and MDA- MB-231 cell lines (90) has found that MCF10A cells were Sodium Butyrate and SAHA. Sodium butyrate (NaB) is shown minimum affected by elevated doses of these HDAC to exhibit beneficial anticancer ability (81). It has the potential inhibitors while the other tested cell lines were extremely to relax the chromatin structure, allowing easier access to sensitive to treatment with both NaB and TSA. Concerning transcription-related proteins. These abilities render NaB a the TNBC cell line (MDA-MB-231), NaB treatment potent treatment against several types of solid tumor (82, 83). significantly enhanced the activity of pyruvate kinase, but it A study in MDA-MB-231 and BT-549 cell lines using various neither induced an attenuation of glycolysis, nor enhanced the concentrations of NaB and SAHA for 24 h revealed that these activity of lactate dehydrogenase, as it happened in T-47 D inhibitors suppress cell proliferation, arrest cell cycle at G 0/G 1 cells. Furthermore, in both T-47D and MDA-MB-231 cells, phase, and finally are involved in mitochondrial-related the rate-limiting enzyme of the pentose phosphate pathway, apoptosis. Moreover, both SAHA and NaB treatment Glucose-6phosphate dehydrogenase (G6PDH) was activated decreased the phosphorylation, and also the mRNA and by NaB treatment, while in MCF-7 cells, G6PDH activity was

304 Garmpis et al : Histone Deacetylase Inhibitors in Triple Negative Breast Cancer (Review) inhibited. Finally, both NaB and TSA in MDA-MB-231 and in hematologic, lung, breast, ovarian, thyroid and prostate T-47D cells significantly raised the oxygen consumption. malignancies (104). Recently panobinostat was approved for These data show that mitochondria might occupy a key role treatment of multiple myeloma with tumor progression after in metastasis (90). Further research is required in order to immunomodulating agents and bortezomib (105). understand the pathways of action of these inhibitors and then Interestingly, a study has reported significant action of target the proper signals to suppress the specific BC type. panobinostat against the TNBC, as it exerted anti-proliferative action, provoked apoptosis and inhibited the primary tumor Mocetinostat. Mocetinostat is a class I HDAC inhibitor that is volumes of TNBC xenografts (106). Also in the same study, associated with the reversion of cardiac fibrosis (91). Recently, in vitro and in vivo results have shown that treatment with mocetinostat was reported to have promising antitumor panobinostat increases significantly the epithelial cell marker activities against various types of cancer cell lines and tumor CDH1. In general, the expression of EMT markers, such as xenografts in nude mice, as it is related to apoptosis (92-94). A epithelial-cadherin (CDH1), neuronal-cadherin (CDH2), study showed that mocetinostat inhibits the growth of colon vimentin (VIM), zinc finger E-box binding homeobox 1 cancer cells via the up-regulation of WNT ligand DKK-1 (ZEB1) and 2 (ZEB2), has been associated with poor expression (95). Also, in B-cell chronic lymphocytic leukemia prognosis of BC patients (107-109). As a result, these markers cells, it has been shown to lead to apoptosis, as it decreases the have become attractive targets for the confrontation of expression of Mcl-1 protein and it induces Bax translocation to metastasis. mitochondria (96). Another study has shown that mocetinostat In a recent study it has been tested whether panobinostat has inhibits the proliferation of prostate cancer cells (93). the ability to inhibit the migration, invasion, and metastasis of Borbely et al. studied the combination of mocetinostat and MDA-MB-231 and BT-549 cell lines, by suppressing EMT JQ1 in MDA-MB-231, BT549, MCF-7 and T47-D cells (97). (110). In TNBC cells, panobinostat has been reported to inhibit JQ1 is a bromodomain and extra-C terminal (BET) inhibitor the expression of genes associated to EMT and to induce the and is reported to participate in the expression of oncogenes expression of CDH1, while these results were not found in and tumor suppressor genes. JQ1 is in pre-clinical testing for luminal A or basal BC cell lines (106). This study has also the treatment of hematological malignancies and reported very important results about panobinostat effects on neuroblastoma (98-100). Researchers found that either JQ1 or MDA-MB-231 and BT-549 cells, as it augmented histone mocetinostat treatment alone, inhibited the expression of genes acetylation, while suppressed the cell proliferation and viability. involved in cell cycle regulation. Interestingly, the It led to cell cycle arrest at G 2/M phase with a simultaneous combination of JQ1 and mocetinostat decreased even more decrease in S phase. Moreover, panobinostat induced apoptosis cell viability as the compounds acted synergistically. at 24 h. In addition, treatment of ER-negative breast cancer Furthermore, combination treatment was associated with a cells (MDA-MB-231 and MDA-MB-435) with panobinostat sharp increase in the expression of many members of the for 24 h resulted in restoration of ER α mRNA and protein ubiquitin–specific protease 17 (USP17) family of expression. However, reactivation of ER α expression was not deubiquitinating enzymes (97). Previous studies have reported due to changes in the methylation profile of CpG island within that USP17 regulates Ras/MAPK signaling partly through the ER promoter region. Interestingly, after at least 96 h from regulation of the RCE1 (101). It is known that the Ras/MAPK termination of panobinostat treatment, the expression of ER α pathway is involved in BC progression (102). The activity of this mRNA was maintained. Panobinostat treatment released DNA pathway has been tested and it was found that the expression of (cytosine-5)-methyltransferase 1 (DNMT1), HDAC1, and the USP17L5 protein was increased, when at the same time Ras H3 lysine 9 (H3-K9) methyltransferase SUV39H1 from the ER activity was decreased. In other words, USP17 enzymes promoter. Investigators claimed that these changes altered the attenuated the Ras/MAPK pathway and cell viability was chromatin state to active, characterized by increased the levels suppressed. On the other hand siRNA-mediated depletion of of acetylated H3 and H4, decreased methylated H3-K9 and USP17, decreased the cytoxicity, indicating that the Ras/MAPK false binding of heterochromatin protein 1 (HP1 alpha) at the signaling pathway is involved in the synergistic effect of the promoter (111). combinational treatment (97). All these data show that the Furthermore, Rhodes et al. compared panobinostat to other combination of HDAC and BET inhibitors could be used as HDAC inhibitors (SAHA, TMP269) and found that potential therapeutic strategy for BC, given that it reduces the panobinostat has much more potent anti-EMT effects than the viability of TNBC cells, through induction of USP17. other inhibitors (110). In vitro research showed that changes in EMT-related gene expression were associated with the Panobinostat. Panobinostat (LBH589) is a pan-HDAC suppression of cell migration and invasion. Also, in a inhibitor. It has the ability to block several pathways related xenograft model these alterations have induced a statistically to cancer and reverse epigenetic events related to cancer significant inhibition of metastasis of TNBC cells to both progression (103). Its anti-cancer effects have been observed brain and lung. Except for CDH1expression, panobinostat has

305 CANCER GENOMICS & PROTEOMICS 14 : 299-313 (2017) induced the suppression of multiple predominant pro-EMT A study on MDA-MB-231cells investigated the in vitro and genes including ZEB1 and ZEB2 . In addition, when in vivo effect of co-treatment of panobinostat with the investigators forced exogenous expression of ZEB1 or ZEB2 , autophagy inhibitor chloroquine (123). First of all, the inhibitory effects of panobinostat on cell migration, panobinostat treatment disrupted the hsp90/HDAC6/HSF1/p97 invasion, and CDH1 expression were reversed (110). complex (124, 125) and induced a heat shock response It has been recently reported that ZEBs recruit Class I mediated by heat shock factor protein 1 (HSF1). This HDACs (48, 112, 113). Given the fact that panobinostat can abruption of the HSF1 from the multiprotein comlex is act against Class I, II and IV HDACs, in addition to inhibition reported to lead to HSF1 phosphorylation, trimerization and of ZEB expression, panobinostat has the ability to prevent nuclear translocation (125, 126). Moreover, panobinostat ZEB-mediated repression of CDH1. treatment induced endoplasmic reticulum stress response and However, it is probably the repression of ZEB2 that autophagy (123), evidence also supported by previous research regulates the effects of panobinostat on tumor genesis, as (127-129). Combinational treatment of panobinostat with ZEB1 expression alone was not sufficient to overcome the chloroquine was found to significantly enhance both the in actions of panobinostat on CDH1 expression (110). To sum vivo and the in vitro effect of panobinostat, given the fact that up, it is critical to study panobinostat as a novel therapeutic to it inhibited tumor growth and increased viability of MDA- target the aggressive and metastatic nature of TNBC, as MB-231 xenografts (123). panobinostat is claimed to inhibit both TNBC primary tumor Therefore, panobinostat could be used for treatment of genesis and the metastasis of TNBC cells. aggressive BC that is resistant to hormonal therapy. We Another study has confirmed these findings, given that it propose that panobinostat should be tested either in was found that in MDA-MB231 cells independent of estrogen monotherapy, or in combination with known anti-cancer receptor expression, panobinostat induced CDH1 expression agents, like trastuzumab, capecitabine, lapatinib, or paclitaxel. (114). Panobinostat was reported to induce the expression of CDH1 and reduce the migration and invasion of TNBC cells. Entinostat. Previous studies have reported that retinoic acid Researchers claimed that panobinostat by-passed the CDH1 and its products, such as all-trans retinoic acid (ATRA), urge transcriptional repressors (Snail, Slug), while the ERa the differentiation of multiple types of stem cells, including expression and pathway were not affected. the stem cells that exist in BC (130, 131). On the other hand, Another combination that has been studied on TNBC cell ATRA has been used in a clinical trial and its efficacy was lines (HCC1937 and MDA-MB-231) is co-treatment of limited (132), possibly because of the epigenetic knock-out of panobinostat and salinomycin (115). Salinomycin is an the retinoic acid receptor (RAR)- β (133). Recent studies have antibiotic that has been used in farm animals (116) and it has shown that HDAC inhibitors have the ability to re-express been associated with the inhibition of BC stem cells in TNBC RAR- β, so they restore the sensitivity of the cells to treatment (117, 118). Also, studies in glioblastoma have shown that co- (134-136). It has been reported that co-treatment of HDAC treatment of salomycin and HDAC inhibitors induced the inhibitors with either doxorubicin or retinoids increased the death of stem-like glioblastoma cells (119). Kai et al. have cytoxicity against cancer cells (75, 137). reported that panobinostat and salinomycin acted A novel study has examined the effects of co-treatment with synergistically and inhibited the TNBC cell proliferation (115). entinostat (MS-275), which is a selective inhibitor of class I HDAC inhibitors have been found to be beneficial in BC HDACs, ATRA, and doxorubicin and reported that TNBC cell therapy, as they target BC stem cells, have anti-proliferative growth was inhibited in xenografts and killed tumor cells and effects on aldehyde dehydrogenase 1 (ALDH1)-positive cells, in cell cultures (138). Given the fact that direct binding of and suppress the formation of BC mammospheres (120). Also, topoisomerase II- β ( TopoII- β) to RAR- β promoter has been ALDH1 has been associated with TNBC, so ALDH1 can be reported in acute pro-myelocytic leukemia cells (139), used as a BC stem cell marker in TNBC (121, 122). Based on investigators have searched that path and found that entinostat these facts, Kai et al. determined the activity of panobinostat and doxorubicin treatment suppressed the expression of Topo on TNBC cells with BC stem cell properties (115). Co- II- β. As a result, RAR- β expression was silenced. It was treatment of panobinostat and salinomycin inhibited the BC demonstrated that this co-treatment achieved significant tumor stem cell properties, as the self-renewal capacity and ALDH1 suppression in TNBC, as entinostat urges the cytoxicity activity were suppressed. Moreover, this combinational mediated by doxorubicin and the differentiation mediated by treatment inhibited growth of ALDH1-positive cells, induced retinoid (138). apoptosis, arrested cell cycle and regulated EMT in BC stem As we mentioned, in metastatic cell lines and invasive BCs cells (115). We can assume that further research is needed, E-cadherin is silenced epigenetically and EMT phenotype is given that co-treatment of panobinostat with salinomycin in reported to be the critical step in the beginning of metastasis TNBC is claimed to provide an effective targeted therapy for (140-142). In MDA-MB-231 and Hs578T cells, entinostat patients with TNBC. treatment has been reported to reverse the EMT phenotype

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(143). These cell lines have lost the expression of E-cadherin, the most promising results between other derivatives that were while they highly express mesenchymal markers such as N- submitted to docking simulations. In vitro studies against cadherin and vimentin along with transcriptional repressors HeLa, rhabdomyosarcoma and BC cells lines have shown that such as Twist and Snail (144-147). Treatment with entinostat compound 2 was the best anticancer agent (154). Interestingly, increased the transcription of E-cadherin, while the mRNA it was effective against TNBC cells. It seemed that compound expression of N-cadherin was decreased. Chromatin 2 targeted HDAC8. In addition, in vitro studies have shown immunoprecipitation analysis demonstrated that E-cadherin’s that compound 2 inhibited cancer cell proliferation at a much promoter was activated due to a decrease in the association of lower concentration, compared to valproic acid (154). TWIST and SNAIL with E-cadherin promoter (143). In a previous study, it has been reported that HDAC1 activity is Uncommon Results required for the SNAIL-mediated repression of E-cadherin (145), so investigators studied the activity of HDAC1 and Unfortunately, despite the promising results in several pre- found that entinostat inhibits HDAC1, thus the repression of clinical trials, when HDAC inhibitors are used as single E-cadherin is reversed. An in vitro study has shown that agents, they failed to be clinically beneficial against solid entinostat inhibited cell migration. Moreover, there was tumors (33). For instance, in the field of metastatic BC, SAHA observed an increase of vimentin phosphorylation levels, as treatment in 14 patients failed to achieve adequate single-agent well as changes in vimentin filaments. Interestingly, the activity (155). Moreover, de Cremoux et al. have used increased phosphorylation of vimentin led to suppression of different HDAC inhibitors (SAHA, panobinostat, abexinostat) the formation of microtentacles based on tubulin (143), which in MDA-MB231, Hs 578T and SUM149 human cell lines and are known to help floating cells attach to other surfaces (148). found that despite the cytotoxicity of both of these inhibitors We should also mention that a subset of cells within a Estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) breast tumor may cause escape from the primary site. These genes were not re-expressed in vitro (156). Xenograft studies cells are named as tumor-initiating cells (TICs). TICs produce with abexinostat treatment for three consecutive days did not progenitor cells, which cannot self-renew, but they may induce the expression of ESR1/ESR1 related genes or of ERa comprise the greater part of the tumor (149). Entinostat protein. In addition, intra-tumor H3 acetylation was observed treatment on TNBC cells has been shown to decrease the level (156). Another study in MDA-MB231 and SKBR3 lines of TICs, therefore entinostat might be used to prevent treated with SAHA or valproic acid for 48 h, showed minimal development of metastasis (150). or no alterations in ERa or in ERb mRNa and protein (157). It is apparent that the ability of entinostat to reverse the Also, different HDAC inhibitors have been shown to cause EMT phenotype and reduce the migration of TNBC cells, as different effects on luminal and mesenchymal-like breast it reduces the attachment of floating cancer cells to new cancer cells, either elevating histone acetylation or showing surfaces, render it a promising therapeutic agent against no effects (158). metastasis. Conclusion YCW1. The octanedioic acid [3-(2-(5-methoxy-1 H-indol- 1yl)ethoxy)phenyl]-amide N-hydroxyamide (YCW1) is a TNBC is a serious subtype of BC that is correlated with poor novel HDAC inhibitor that has been developped by structure- prognosis due to the high proliferation rate. New therapeutic based analyses (151, 152). Researchers have tested the co- strategies are needed in order to cure TNBC. Epigenetic treatment with YCW1 and ionizing radiation (IR) in a murine mutations are associated with repression of BC development and a human TNBC cell line, 4 T1, and MDA-MB-231, and therapy resistance. respectively. This combination was shown to increase Between all the epigenetic treatments, HDAC inhibitors autophagy and endoplasmic reticulum stress, so these represent the first successful anti-cancer epigenetic therapy. compounds acted as a cytotoxic. Interistingly, in comparison Studies show that they affect positively the therapy of to the effects of SAHA, YCW1 significantly enhanced subtypes of hematological malignancies. However, it is still toxicity. Study in an orthotopic BC mouse model confirmed unclear how effective they are against solid tumors (159). these results (153). Therefore, co-treatment with IR and Nevertheless, the majority of studies in the field of TNBC YCW1 induced autophagic cell death. therapy tend to combine HDAC inhibitors with kinase inhibitors, autophagy inhibitors, antibiotics, chemotherapy, IR, N-(2-Hydroxyphenyl)-2propylpentanamide. N -(2-Hydroxyphe- or even two HDAC inhibitors treatment in order to enhance nyl)-2propylpentanamide (Compound 2) is an aryl derivative their efficacy against TNBC (47, 69, 71, 84, 115, 123, 153). of valproic acid. These derivatives are produced by combining In the majority of the studies, co-treatment of an HDAC valproic acid and the arylamine core of SAHA with different inhibitor with another compound induced the inhibition of substituents at its carboxyl group. Compound 2 has exerted tumor growth and showed anti-proliferative effects (46, 47,

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77, 84, 115, 123, 138, 154). Also, several HDAC inhibitors Lippman M, Ethier S, Gazdar A and Gray JW: A collection of are assosiated not only with autophagic cell death, but also breast cancer cell lines for the study of functionally distinct apoptosis (44, 55, 70, 71, 84, 97, 153). Interestingly, many cancer subtypes. Cancer Cell 10 : 515-527, 2006. 9 Bernhardt SM, Dasari P, Walsh D, Townsend AR, Price TJ and studies have found that HDAC inhibitors have the ability to Ingman WV: Hormonal Modulation of Breast Cancer Gene convert ER-negative tumors to ER-positive tumors (111, 160, Expression: Implications for Intrinsic Subtyping in 161). Finally, a recent study showed that entinostat treatment Premenopausal Women. Front Oncol 6: 241, 2016. can prevent the metastasis (143). 10 Mohamed A, Krajewski K, Cakar B and Ma CX: Targeted However, as we mentioned above, we should not forget that therapy for breast cancer. Am J Pathol 183 : 1096-1112, 2013. there are several studies in the pertinent literature with 11 Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz contradictory results (155-157). These unexpected studies JI, He X and Perou CM: Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast reveal the necessity for further investigation in the field of cancer. Breast Cancer Res 12 : R68, 2010. HDAC inhibitors against TNBC cancer. We should understand 12 Marks DL, Olson RL and Fernandez-Zapico ME: Epigenetic the nature of the molecular basis of the selectivity of HDAC control of the tumor microenvironment. Epigenomics 8: 671- inhibitors and which are the proper combining treatments in 1687, 2016. order to find more clinically beneficial treatment against 13 Jones P: The cancer epigenome. Genome 56 : 540-541, 2013. TNBC. 14 Rando OJ and Ahmad K: Rules and regulation in the primary structure of chromatin. Curr Opin Cell Biol 19 : 250-256, 2007. 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